CN116262922A

CN116262922A - Application of potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes

Info

Publication number: CN116262922A
Application number: CN202111525533.8A
Authority: CN
Inventors: 孟赞; 李清清; 王庆国; 李广存; 刘士扬; 张苗苗
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2023-06-16

Abstract

The invention discloses an application of a potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes, and belongs to the technical field of molecular genetics. The nucleotide sequence of StSAUR30231 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO. 2. According to the invention, the StSAUR30231 gene is connected to an expression vector, and agrobacterium tumefaciens is utilized to infect and transform the potato, and the data show that the anti-browning capability of the potato can be obviously improved by over-expressing the StSAUR30231 gene. Meanwhile, a homozygous mutant of the gene is obtained through a CRISPR-Cas9 technology, and the result shows that the over-expression StSAUR30231 remarkably reduces the browning of the fresh-cut potatoes, and the anti-browning capability of the mutant is remarkably lower than that of a wild type. The StSAUR30231 gene provided by the invention can provide theoretical basis for cultivating new varieties of good crops, and has great significance in production practice.

Description

Application of potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes

Technical Field

The invention relates to the technical field of molecular genetics, in particular to application of a potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Potatoes are the fourth largest food crop in the world, rich in nutrition, and are known as "nutrient king" (Millam, 2006; tao et al, 2021). With the promotion of the strategy of potato staple food conversion in China, more and more potatoes are on dining tables of people. Fresh-cut potatoes are increasingly favored by consumers because of their convenience, freshness, nutrition, etc. (Wu, 2019). However, potatoes are susceptible to enzymatic browning after cutting, which severely reduces the organoleptic quality and commercial value of freshly cut potatoes (mosneakuta et al 2012; zhu et al 2020).

In recent years, genetic engineering technology is rapidly developed, and genetic engineering has important significance for the development of future agricultural science. The gene technology is used to improve the characteristics of potato, so that the method is an effective means for improving the quality of potato.

Transcription is possible within minutes under auxin induction, and this induction does not require the synthesis of new proteins, such genes being termed early or incipient auxin response genes (Abel and Theologis, 1996). The SAUR gene family is the first early auxin response gene identified (McClure and Guilfoyle, 1987). In plants, some SAUR genes may be involved in promoting plant growth (Li et al, 2015), promoting cell elongation (Spartz et al, 2012), modulating top hook development (Park et al, 2012), and some SAUR genes negatively regulate auxin synthesis and transport (Kant et al, 2009), promoting plant senescence, etc. (Kant et al, 2009; hou et al, 2013; wen et al, 2020). However, to date, no report has been made on the effect of the SAUR gene on potato browning.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide an application of a potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes. According to the research of the invention, the SAUR gene StSAUR30231 of the potato is closely related to the browning resistance of the potato, and the overexpression of the StSAUR30231 gene can obviously reduce the browning of the fresh-cut potato; and the anti-browning capability of the StSAUR30231 gene mutant is obviously lower than that of the wild type. Therefore, the StSAUR30231 gene can provide theoretical basis for cultivating new varieties of good crops, and has great significance in production practice.

The invention is realized by the following technical scheme:

in a first aspect of the invention, there is provided the use of a StSAUR30231 gene for inhibiting enzymatic browning of fresh cut potatoes; the StSAUR30231 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule which has 90% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. Wherein, the cDNA sequence of StSAUR30231 gene is shown in SEQ ID NO. 1; the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed using computer software, for example, using the BLAST algorithm (Altschul et al 1990.Journal of Molecular Biology215:403-410;Karlin and Altschul.1993.Proceedings of the National Academy of Sciences90:5873-5877).

In a second aspect, the invention provides an application of a protein coded by a StSAUR30231 gene in inhibiting enzymatic browning of fresh-cut potatoes; the protein is shown in any one of the following (A1) or (A2):

(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;

(A2) A fusion protein obtained by ligating the N-terminus and/or C-terminus of the protein defined in (A1) with a tag.

Wherein, the proteins (A1) and (A2) can be synthesized artificially or can be obtained by synthesizing the encoding genes and then biologically expressing.

In order to facilitate purification of the protein of (A1), a tag may be attached to the amino-terminal or carboxyl-terminal end of the protein of (A1). The tag may be Poly-Arg (typically 6 RRRRRs), poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

In a third aspect, the invention provides an application of a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium containing the StSAUR30231 gene in inhibiting enzymatic browning of fresh-cut potatoes.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises binary agrobacterium vectors, vectors which can be used for plant microprojectile bombardment, and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant expression vectors. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide thereof, and can be used alone or in combination with other plant promoters; in addition, when the recombinant expression vector is constructed using the gene of the present invention, enhancers including translational enhancers or transcriptional enhancers may also be used.

In a fourth aspect, the present invention provides the above-mentioned StSAUR30231 gene, a protein encoded by the StSAUR30231 gene, a recombinant expression vector containing the StSAUR30231 gene, a transgenic cell line or a genetically engineered bacterium, for use in any one of the following (1) or (2):

(1) Plant breeding;

(2) Regulating and controlling the anti-browning capability of the plants.

In the above application, the plant is preferably potato.

In a fifth aspect of the invention, there is provided a method of inhibiting enzymatic browning of a fresh-cut potato comprising: and (3) overexpression of StSAUR30231 gene in potato.

In the method, the StSAUR30231 gene in the potato can be over-expressed by exogenous transfer into the StSAUR30231 gene; or up-regulate expression of the StSAUR30231 gene in the potato genome. Wherein the method of up-regulating StSAUR30231 gene expression in potato genome comprises: introducing a DNA fragment capable of activating or increasing the transcription level or translation level or protein activity of the StSAUR30231 gene; or control the synthesis of specific small RNA molecules, up-regulate the accumulation of mRNA of StSAUR30231 gene.

The specific small RNA molecules include: microRNA molecules (micrornas, mirnas), interfering micrornas (small interfering RNAs, sirnas), or artificial mirnas (artifical microRNA, amiRNA).

In a sixth aspect of the present invention, there is provided a method for cultivating potatoes having improved anti-browning activity, comprising the steps of:

transferring StSAUR30231 genes into potato original plants, and enabling the StSAUR30231 genes to be over-expressed to obtain potato transgenic plants; the brown-out resistance of the transgenic potato plant is higher than that of the original potato plant.

In the cultivation method, the method for transferring the StSAUR30231 gene into the potato starting plant comprises the following steps: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.

In a seventh aspect of the present invention, there is provided a method for cultivating potatoes having reduced anti-browning ability, comprising the steps of: inhibiting the expression of StSAUR30231 genes in potato genome, and screening to obtain potato plants with reduced anti-browning capability.

In the above method, the method for inhibiting expression of StSAUR30231 gene in potato genome comprises: mutating or knocking out all or part of the sequence of StSAUR30231 gene in potato genome; or interfering with expression of the StSAUR30231 gene using interfering RNA; alternatively, the StSAUR30231 gene is silenced using a gene silencing system.

In an eighth aspect of the present invention, there is provided a method for obtaining a potato plant carrying the StSAUR30231 gene described above, comprising the steps of:

obtaining plant cells containing the StSAUR30231 gene by means of transgenesis or genome editing; regenerating the obtained plant cells into seedlings.

The invention has the beneficial effects that:

according to the invention, the potato auxin response gene StSAUR30231 is obtained by first cloning, and is transferred into the potato by an agrobacterium-mediated potato transformation method, and analysis proves that the browning degree of the transgenic potato is obviously reduced after the StSAUR30231 gene is overexpressed. The invention explains the biological significance of the potato gene StSAUR30231, provides available data for quality improvement of other crops, and has great significance in production practice.

Drawings

FIG. 1 (a) shows browning of potatoes cut for 3 days with Kexin No. 4 (K4) and Kexin No.13 (K13); FIG. 1 (b) shows changes in the expression level of StSAUR30231 gene 3 days after cutting of K4 and K13 potatoes.

FIG. 2 (a) is an integrated PCR identification of the overexpressed potato StSAUR30231 gene; FIG. 2 (b) shows the measurement of the expression level of the overexpressed potato StSAUR 30231.

FIG. 3 (a) is a PCR identification of a CRISPR knockout potato strain of the exogenous gene StSAUR 30231; FIG. 3 (b) is a second generation sequencing of the CRISPR knockout potato line of the exogenous gene StSAUR 30231.

FIG. 4 (a) is a transgenic potato fresh cut browning phenotype; fig. 4 (b) is a transgenic potato L x value.

FIG. 5 (a) is a transgenic Potato Polyphenol Oxidase (PPO) activity; FIG. 5 (b) shows the expression level of PPO gene.

FIG. 6 is a graph showing the tyrosine content of transgenic potatoes.

FIG. 7 (a) is the radical scavenging of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH); FIG. 7 (b) shows Peroxidase (POD) activity; FIG. 7 (c) shows the Malondialdehyde (MDA) content.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As previously mentioned, potatoes are susceptible to enzymatic browning after cutting, which severely reduces the organoleptic quality and commercial value of freshly cut potatoes. At present, few reports for improving the browning resistance of potatoes by using genetic engineering are available.

In view of the above, the invention provides a SAUR gene StSAUR30231 of potato, wherein after the gene is edited by a CRISPR-Cas system in a fixed point manner, the browning degree of the potato is enhanced, and the anti-browning capability of the potato is remarkably improved after the gene is overexpressed.

According to the invention, stSAUR30231 genes are screened according to transcriptome sequencing (RNA-seq) of gram-new No. 4 (potato easy to brown) and gram-new No.13 (potato difficult to brown), full-length cDNA of the StSAUR30231 genes is cloned from tetraploid Desiree potato, and functional verification is carried out in the potato. The present invention has been made in view of the above.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers. Wherein:

the potato materials used in the invention, namely potato gram-new No. 4 and potato gram-new No.13, and Desiree' potato, are all existing potato varieties. The public is also available from the applicant within 20 years from the date of filing for use in repeating the present invention.

Example 1: method for obtaining anti-browning gene of potato

According to the invention, the potato gram number 4 (K4) which is easy to brown and the potato gram number 13 (K13) which is difficult to brown are selected as experimental materials (figure 1 a), and are freshly cut, stored at 4 ℃, sampled for 0 day and 3 day after cutting, and quick frozen in liquid nitrogen. Total RNA of the above samples was extracted according to the RNA extraction kit from Kangji corporation and sent to Huada genes for transcription sequencing. Transcriptome data analysis found that 121 differential genes were not expressed or expressed in unchanged amounts in the Kexin No. 4 potato but significantly upregulated in the Kexin No.13 potato, 3 days after cutting compared to 0 days after cutting. The genes were subjected to GO enrichment analysis, screened for "hormone response" pathways, and studied from these to StSAUR30231 with higher fold difference (fc=5.8) as candidate genes, and the expression pattern of this gene was again determined by qPCR (fig. 1 b), confirming that the gene expression level was significantly higher in the non-browning prone variety K13 than in the browning prone variety K4, we speculated that the StSAUR30231 gene might be involved in enzymatic browning of potatoes.

qPCR reactions, primers were as follows:

StActin-F：CCTGTTCTACTCACCGAAGCACCTC；(SEQ ID NO.3)

StActin-R：AGCATATCCCTCATAGATTGGGACA。(SEQ ID NO.4)

StSAUR30231-F1：GGGTTGTCTTCCAGTATTGGTA；(SEQ ID NO.5)

StSAUR30231-R1：GTGGCATGAGTTTCTTTGTCAA。(SEQ ID NO.6)

the qPCR reaction system and procedure are shown in Table 1.

Table 1: qPCR reaction system

Example 2: cloning of StSAUR30231 Gene sequence

(1) Primer sequences

According to the CDS sequence of potato genome library (http:// spuddb. Uga. Edu /), the StSAUR30231 gene (PGSC 0003DMG 400030231) is searched, and the primer is designed to amplify the CDS full-length sequence of the gene, wherein the primer sequence is as follows:

StSAUR30231-F2:

5’-gagaacacgggggactctagaATGGGTAGTGGAGATCACAAACACC-3’；(SEQ ID NO.7)

StSAUR30231-R2:5’-ggactgaccacccggggatccGGCCTTGTAGCACCAAGCAT-3’。(SEQ ID NO.8)

(2) RNA extraction

A sample of "Desiree" potato tubers was ground to a powder in liquid nitrogen and total RNA was extracted using the OmniPlant RNA Kit (DNase I) kit from century Corp:

1) Homogenizing: taking 50-100mg plant tissue, rapidly grinding into powder in liquid nitrogen, adding 500 μl Buffer RLS (before use, checking whether β -mercaptoethanol is added), immediately vortexing, and shaking thoroughly.

2) Centrifuge at 12,000rpm (13,400 Xg) at 4℃for 2min.

3) Transferring the supernatant to a filter column (Spin Columns FS) loaded into a collection tube, centrifuging at 12,000rpm at 4deg.C for 1min, carefully sucking the supernatant from the collection tube and transferring to a new RNase-Free centrifuge tube (self-contained), and avoiding the gun head from contacting the cell debris precipitation in the collection tube as much as possible.

4) Slowly adding 0.5 times of absolute ethyl alcohol, mixing, and transferring the obtained solution and precipitate into adsorption column (Spin Columns RM) filled into collecting tube, if all the solution cannot be added into the adsorption column at one time, transferring into the adsorption column twice. Centrifuging at 12,000rpm at 4deg.C for 1min, discarding the waste liquid, and placing the adsorption column back into the collection tube.

5) Adding 350 μl Buffer RW1 into the adsorption column RM, centrifuging at 12,000rpm at 4deg.C for 1min, discarding the waste liquid, and placing the adsorption column back into the collection tube.

6) Preparing DNase I mixed solution: mu.L of RNase-Free Water was taken, and 8. Mu.L of 10 Xreaction Buffer and 20. Mu.L of DNase I (1U/. Mu.L) were added thereto, followed by mixing to prepare a Reaction solution having a final volume of 80. Mu.L.

7) Directly adding 80 μl of DNase I mixture into the adsorption column, and incubating at 20-30deg.C for 15min.

8) Adding 350 μl Buffer RW1 into the adsorption column RM, centrifuging at 12,000rpm at 4deg.C for 1min, discarding the waste liquid, and placing the adsorption column back into the collection tube.

9) 500 μL Buffer RW2 (checked before use to see if absolute ethanol is added) was added to the column RM, centrifuged at 12,000rpm for 1min at 4deg.C, the waste liquid was discarded, and the column was returned to the collection tube.

10 Repeating step 9).

11 Centrifugation at 12,000rpm at 4℃for 2 min).

12 Loading the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 mL), suspending and dripping 30-50 μl RNase-Free Water into the middle part of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12,000rpm at 4deg.C for 1min, and storing the obtained RNA solution at-70deg.C to prevent degradation.

(3) Reverse transcription (cDNA first Strand Synthesis)

Reverse transcription was performed on the above sample RNA using the HiFiScript cDNA Synthesis Kit kit of century, the system was shown in Table 2, and after mixing, the sample was incubated at 42℃for 15min, at 85℃for 5min, centrifuged briefly, and cooled on ice. The product was used for subsequent gene cloning.

Table 2: cDNA first strand synthesis reaction system

(4) PCR amplification of target Gene StSAUR30231

Using the cDNA obtained as described above as a template and StSAUR30231-F2 (SEQ ID NO. 7) and StSAUR30231-R2 (SEQ ID NO. 8) as amplification primers, a PCR reaction system shown in Table 3 was prepared, and the CDS sequence (390 bp) of StSAUR30231 was cloned. The PCR reaction procedure was 98℃for 2min;98 ℃ for 10s;98 ℃ for 30s; cycling for 35 times at 72 ℃ for 30 seconds; 72℃for 10min.

Table 3: PCR reaction system

The PCR amplified products were detected by 1% agarose gel electrophoresis, and then the target bands were recovered by gel cutting using the full gold Quick Gel Extraction Kit kit.

Example 3: construction of plant expression vector PBI-StSAUR30231

The expression vector PBI121 was linearized with XbaI and BamH1 restriction enzymes, and then the target fragment obtained by gel recovery was ligated with the linearized vector PBI121 using ClonExpress II One Step Cloning Kit homologous recombinase from Norvezan, the ligation system was as shown in Table 4, mixed well, ligated for 30min at 37℃and placed on ice for cooling and stored at-20 ℃.

Table 4: homologous recombination connection system

Table 4 Reaction mixture of homologous recombination linkage

The ligation product was then transformed into E.coli DH 5. Alpha. After the strain grows out, selecting a monoclonal, culturing at 37 ℃ at 200rpm for overnight, then carrying out PCR identification by using corresponding primers, selecting bacterial liquid of positive clones, sequencing by a sequencing company (a biological organism), carrying out plasmid extraction on the strain with correct sequencing, and then carrying out heat shock conversion on the recombinant plasmid to agrobacterium AGL1+VirG.

(5) And selecting a monoclonal to carry out bacterial liquid PCR identification, and obtaining the recombinant plasmid for successfully transforming the agrobacterium.

Example 4: construction of CRISPR Cas9 knockout vector of StSAUR30231 gene

CRISPR vector pCAMBIA1301 is used as a basic vector, CRISPR-P2.0%http:// Crispr.hzau.edu.cn/CRISPR2 /) was designed as follows 2The expression vector was constructed by ligating g1 and g2, g3 and g4 to two pCAMBIA1301 vectors, respectively.

g1:CCATCAACACGGTCATGGGATAA，(SEQ ID NO.9)

g2:GAAATGATAGGCATCCCAAAGGG；(SEQ ID NO.10)

g3:AAAGGGTTGTCTTCCAGTATTGG，(SEQ ID NO.11)

g4:CCAGTATTGGTAGGTCATGATGG。(SEQ ID NO.12)

Example 5: genetic transformation of potato

(1) Potato transformation medium

The medium was prepared by the method described in Li et al (Li et al, 2016). M1 medium: MS+0.2mg/L NAA (a-naphthylacetic acid) +0.02mg/L GA3 (gibberellin) +2.5mg/L ZT (zeatin); m2: m1 medium+100 mg/LTim (timentin) +50mg/L Kan (kanamycin); m3 medium: MS+0.02mg/L NAA (a-naphthylacetic acid) +0.02mg/L GA3 (gibberellin) +2.0mg/L ZT (zeatin) +100mg/L Tim (timentin) +50mg/L Kan (kanamycin); m4 medium: MS+100mg/L Tim (timentin) +50mg/L Kan (kanamycin).

(2) Genetic transformation of potato

Genetic transformation of potatoes is described in reference to Li et al (Li et al 2016). Selecting vigorous Desiree potato seedlings, cutting the stem sections into 0.5cm explants, and culturing in Agrobacterium (OD) ₆₀₀ =0.6-0.8) for 20min, and then placed in M1 medium for 2d in the dark at 28 ℃. Then transferring the explant into M2 culture medium, culturing in a climatic chamber with the illumination intensity of 1500lux and the illumination period of 16h/8h at 23 ℃ for 10-12d, transferring the callus with good growth vigor into M3 culture medium, culturing for about 1 month (transferring the stem segments into new M3 culture medium every 2 weeks during the period), cutting off the regenerated buds, and transferring into M4 culture medium.

(3) Planting of potatoes

Rooting strains which over express and knock out StSAUR30231 genes are planted in sterile soil and are cultured in a tissue culture room (the temperature is 23 ℃, the humidity is 70%, the illumination intensity is 1500 lux), after the rooting strains grow for 5 months, the rooting strains are harvested when leaves turn yellow and wither (the commercial maturity is reached).

Example 6: positive identification of transgenic potatoes

1. Positive identification of over-expressed StSAUR30231 potato

1.1 overexpression Potato StSAUR30231 Gene integration PCR identification

(1) Potato leaf genomic DNA was extracted using NuClean Plant Genomic DNA Kit from well-known century corporation, as follows:

1) About 100mg or about 20mg of fresh tissue of the plant is taken, added with liquid nitrogen and fully ground.

2) The milled powder was collected in a centrifuge tube (self-contained), 400. Mu.L Buffer LP1 and 6. Mu.L RNase A (10 mg/mL) were added, vortexed for 1min, and left at room temperature for 10min for complete lysis.

3) 130. Mu.L Buffer LP2 was added, mixed well and vortexed for 1min.

4) Centrifuge at 12,000rpm (13,400 Xg) for 5min and transfer the supernatant to a new centrifuge tube (self-contained).

5) 1.5 volumes of Buffer LP3 (checked before use if absolute ethanol has been added) were added and mixed well (e.g., 500. Mu.L filtrate added 750. Mu.L Buffer LP 3).

6) Adding all the solution and precipitate into adsorption column loaded into collecting tube, centrifuging at 12000rpm for 1min, pouring out waste liquid from collecting tube, and placing the adsorption column into collecting tube again.

7) 500. Mu.L Buffer GW2 is added into the adsorption column, the mixture is centrifuged for 1min at 12000rpm, the waste liquid in the collecting pipe is poured out, and the adsorption column is replaced into the collecting pipe.

8) And 7, repeating the step 7.

9) Centrifuge at 12000rpm for 2min, pour out the waste liquid in the collection tube. The column was left at room temperature for several minutes to dry thoroughly.

10 Placing the adsorption column into a new centrifuge tube, suspending and dripping 50-100 mu L Buffer GE into the middle part of the adsorption film, standing for 2-5min at room temperature, centrifuging at 12,000rpm for 1min, and collecting DNA solution. The DNA was stored at-20 ℃.

(2) The extracted wild type and overexpressed potato DNA were subjected to PCR assays with StSAUR30231-F2 and StSAUR30231-R2 as primers (as described in example 1) and the PCR results were shown in FIG. 2 a. Recombinant plasmid and wild-type (WT) genomic DNA served as Positive Control (PC) and Negative Control (NC), respectively.

1.2 measurement of expression level of overexpressed potato tuber StSAUR30231

Wild-type and overexpressed potato tuber RNA was extracted, reverse transcribed into cDNA, and then the expression level of StSAUR30231 was detected by fluorescent quantitative PCR using UltraSYBRMixture well known as century Co (FIG. 2 b), the reaction system being shown in Table 1, and the reaction procedure was as follows: 95 ℃ for 10min;95 ℃ for 15s;60 ℃,1min,38 cycles; 95 ℃ for 15s;60 ℃ for 1min;95 ℃ for 15s;60℃for 15s. The StActin-F and StActin-R genes are internal genes of potato, and the primer sequences are as described in example 1.

As can be seen from FIG. 2b, the StSAUR30231 gene overexpressing potato lines 0E1 and OE2 tubers was 4-5 fold over-expressed compared to the wild-type.

2. Detection of CRISPR knockout potato lines of StSAUR30231

(1) PCR detection

The rooted potato leaf DNA was extracted according to the method of DNA extraction in 1.1 and the rooted potato seedlings were identified positively by PCR using the following primers. cas9-F GTTCATCAAGCCGATTCTGG (SEQ ID NO. 13); cas9-R GCTTCCTGCTCAGCCTCCC (SEQ ID NO. 14). The recombinant plasmid and wild-type (WT) genomic DNA served as Positive Control (PC) and Negative Control (NC), respectively, and the detection results were as shown in fig. 3 a.

(2) Second generation sequencing

The positive lines were subjected to second generation sequencing with the following primers to detect the mutation type. StSAUR30231-F3: TTACCATTCAACAACACCCTCAAA (SEQ ID NO. 15); stSAUR30231-R3: ACCAAGCATGGTGGTTATGGT (SEQ ID NO. 16). As a result of the second generation sequencing (FIG. 3 b), it was found that both m1 and m2 were homozygous mutants, and that m2 was a frameshift mutant.

3. Characterization of browning phenotype of potato tubers overexpressing and knocked out StSAUR30231 genes

3.1 transgenic Potato browning phenotype verification

And (3) carrying out fresh cutting treatment on the harvested wild tubers and transgenic tubers, sampling a part of the tubers, carrying out physiological index measurement, storing a part of the tubers in a refrigerator at 4 ℃, and observing the browning degree of the tubers. As can be seen from FIGS. 4a-b, the browning level of potatoes (OE 1, OE 2) was significantly reduced after overexpression of the StSAUR30231 gene compared to Wild Type (WT), whereas the knockout lines (m 1, m 2) exhibited a browning-aggravating phenotype.

3.2 determination of physiological index related to browning of transgenic Potato

Polyphenol Oxidase (PPO) is a key enzyme for enzymatic browning of potatoes (Queiroz et al, 2008), tyrosine is a major substrate for enzymatic browning of potatoes (golyer and pelles, 2018), and we have thus determined the PPO activity of transgenic potatoes and their gene expression and tyrosine content. The procedure of PPO activity assay reference Dong et al (2015) was slightly modified; the tyrosine content determination is described in Meng et al (2021). The result shows that after the StSAUR30231 is overexpressed, the PPO activity of potato tubers is reduced, the PPO gene expression is reduced, and the tyrosine content is reduced; whereas the knockdown lines had increased PPO activity and gene expression and increased tyrosine content (FIGS. 5a-b; FIG. 6).

The antioxidant capacity is also an important factor affecting the browning and quality of fresh cut fruits and vegetables (Toivonen and Brummell, 2008). We therefore determined the DPPH radical scavenging rate, POD activity and MDA content of transgenic potato tubers. The method of measuring DPPH radical scavenging rate was slightly modified with reference to the method of Kenny and O' Beirne (2010), POD activity was slightly modified with reference to the method of Yang et al (2009), and MDA content was measured with reference to the method of Barman et al (2014). The results show that: overexpression (mutation) of StSAUR30231 significantly increased (reduced) DPPH radical scavenging and POD activity in potato, decreased (increased) MDA content, decreased (increased) membrane lipid peroxidation levels (fig. 7 a-c).

In conclusion, the StSAUR30231 gene can remarkably inhibit browning of fresh-cut potatoes, and the mechanism of the StSAUR30231 gene comprises the steps of reducing PPO activity and gene expression thereof, reducing tyrosine content and improving oxidation resistance.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong agricultural university

Application of <120> potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh cut potatoes

<130> 2021

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 390

<212> DNA

<213> Potato (Solanum tuberosum L.)

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atgggtagtg gagatcacaa acaccaccat caccatttga atttgcatgt tcaagtgcat 60

ctacctcaca tccattttca ccatcaccat caccatcaac acggtcatgg gataaaagaa 120

atgataggca tcccaaaggg ttgtcttcca gtattggtag gtcatgatgg tgaggaacaa 180

cacaagttca taatcccagt gatatacatt aaccatccac ttttcacaca attgttgaaa 240

ggtaatgaag aagagtgtga acttcatcat gatggtccta tgaatatccc ttgtcatatc 300

gaagaatttc gatacgttga aggtatgatt gacaaagaaa ctcatgccac cggacaccat 360

aaccaccatg cttggtgcta caaggcctga 390

<210> 2

<211> 129

<212> PRT

<213> Potato (Solanum tuberosum L.)

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Met Gly Ser Gly Asp His Lys His His His His His Leu Asn Leu His

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Val Gln Val His Leu Pro His Ile His Phe His His His His His His

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Gln His Gly His Gly Ile Lys Glu Met Ile Gly Ile Pro Lys Gly Cys

35 40 45

Leu Pro Val Leu Val Gly His Asp Gly Glu Glu Gln His Lys Phe Ile

50 55 60

Ile Pro Val Ile Tyr Ile Asn His Pro Leu Phe Thr Gln Leu Leu Lys

65 70 75 80

Gly Asn Glu Glu Glu Cys Glu Leu His His Asp Gly Pro Met Asn Ile

85 90 95

Pro Cys His Ile Glu Glu Phe Arg Tyr Val Glu Gly Met Ile Asp Lys

100 105 110

Glu Thr His Ala Thr Gly His His Asn His His Ala Trp Cys Tyr Lys

115 120 125

Ala

<210> 3

<211> 25

<212> DNA

<213> artificial sequence

<400> 3

cctgttctac tcaccgaagc acctc 25

<210> 4

<211> 25

<212> DNA

<213> artificial sequence

<400> 4

agcatatccc tcatagattg ggaca 25

<210> 5

<211> 22

<212> DNA

<213> artificial sequence

<400> 5

gggttgtctt ccagtattgg ta 22

<210> 6

<211> 22

<212> DNA

<213> artificial sequence

<400> 6

gtggcatgag tttctttgtc aa 22

<210> 7

<211> 46

<212> DNA

<213> artificial sequence

<400> 7

gagaacacgg gggactctag aatgggtagt ggagatcaca aacacc 46

<210> 8

<211> 41

<212> DNA

<213> artificial sequence

<400> 8

ggactgacca cccggggatc cggccttgta gcaccaagca t 41

<210> 9

<211> 23

<212> DNA

<213> artificial sequence

<400> 9

ccatcaacac ggtcatggga taa 23

<210> 10

<211> 23

<212> DNA

<213> artificial sequence

<400> 10

gaaatgatag gcatcccaaa ggg 23

<210> 11

<211> 23

<212> DNA

<213> artificial sequence

<400> 11

aaagggttgt cttccagtat tgg 23

<210> 12

<211> 23

<212> DNA

<213> artificial sequence

<400> 12

ccagtattgg taggtcatga tgg 23

<210> 13

<211> 20

<212> DNA

<213> artificial sequence

<400> 13

gttcatcaag ccgattctgg 20

<210> 14

<211> 19

<212> DNA

<213> artificial sequence

<400> 14

gcttcctgct cagcctccc 19

<210> 15

<211> 24

<212> DNA

<213> artificial sequence

<400> 15

ttaccattca acaacaccct caaa 24

<210> 16

<211> 21

<212> DNA

<213> artificial sequence

<400> 16

accaagcatg gtggttatgg t 21

Claims

Application of StSAUR30231 gene in inhibiting enzymatic browning of fresh cut potatoes; the StSAUR30231 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule which has 90% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
Application of StSAUR30231 gene coded protein in inhibiting enzymatic browning of fresh cut potatoes; the protein is shown in any one of the following (A1) or (A2):

(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;

(A2) A fusion protein obtained by ligating the N-terminus and/or C-terminus of the protein defined in (A1) with a tag.
3. Application of recombinant expression vector containing StSAUR30231 gene, transgenic cell line or genetic engineering bacteria in inhibiting enzymatic browning of fresh cut potato;

the StSAUR30231 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule which has 90% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
StSAUR30231 gene, protein encoded by StSAUR30231 gene, recombinant expression vector containing StSAUR30231 gene, transgenic cell line or genetically engineered bacterium, and the use thereof in any one of the following (1) or (2):

(1) Plant breeding;

(2) Regulating and controlling the anti-browning capability of the plants.
5. The use according to claim 4, wherein the plant is potato.
6. A method of inhibiting enzymatic browning of a freshly cut potato comprising: a step of overexpressing the StSAUR30231 gene in potato;

the StSAUR30231 gene is a nucleic acid molecule as shown in the following i) or ii) or iii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule which has 90% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
7. A method for cultivating potatoes with improved anti-browning capability, comprising the steps of:

transferring StSAUR30231 genes into potato original plants, and enabling the StSAUR30231 genes to be over-expressed to obtain potato transgenic plants; the brown-out resistance of the transgenic potato plant is higher than that of the original potato plant.
8. The method of claim 7, wherein the step of transferring the StSAUR30231 gene into the potato starting plant comprises: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.
9. A method of growing potatoes having reduced resistance to browning comprising the steps of: inhibiting the expression of StSAUR30231 genes in potato genome, and screening to obtain potato plants with reduced anti-browning capability.
10. A method of obtaining a potato plant carrying a StSAUR30231 gene comprising the steps of:

obtaining plant cells containing the StSAUR30231 gene by means of transgenesis or genome editing; regenerating the obtained plant cells into seedlings.